Identification And Functional Study Of Breast Cancer Metastasis Ralated Genes

Objective: To identify the genes related to breast cancer metastasis in order to find the new molecular diagnostic markers and therapeutic molecular targets, and to provide clues for understanding the mechanism of carcinogenisis and metastasis of breast cancer.Methods: The differentially expressed genes between primary breast cancer and paired lymph node metastasis tissues were identified using mRNA differential display, and validated by using reverse dot blot hybridization and cDNA microarray methods. Kinesin-like 4 (KNSL4) mRNA levels in 30 normal breast tissues and in 142 primary breast cancer tissues from the patients with more than five years follow-up were quantified with real time RT-PCR respectively, and the correlations of KNSL4 mRNA level to clinical clinicopathologic factors and prognosis were analyzed. The cellular localization and expression level of Kid protein coded by KNSL4 in breast cancer tissues were detected by immunohistochemistry staining. Furthermore, the pSilencerTM3.1-KNSL4 siRNA recombinant plasmid was constructed and transfected into the breast cancer cell line MDA-MB-435S to establishe the stable sub-cell line of silenced KNSL4 expression, in order to observe the influence of cell biological behavior when KNSL4 expression was down-regulated. Kid regulated target genes were profiled based on the chromatin immunoprecipitation in combination with promoter microarray (ChIP-on-chip) method. The candidate target genes were confirmed by ChIP-qPCR. The mRNA expression of candidate Kid's target genes in MDA-MB-435S/KNSL4 siRNA cells compare with their parental cells were assayed by real time RT-PCR.Result: Four unknown gene fragments were cloned and accepted by the Genbank, with the accession numbers as BG518428, BG518429, BM005520, and BM005521, respectively. The kinesin-like 4 (KNSL4) gene expression was down-regulated in lymph node metastasis in comparison to the paired primary tissues. KNSL4 mRNA was highly expressed in breast cancers, but unexpressed in normal breast tissues (P=0.000). However, lower expression of KNSL4 mRNA in primary cancers correlated to a reduced five years disease-free survival (DFS) of breast cancer patients (P=0.016). KNSL4 mRNA level is prognostic predictor for breast cancer patients in stage I~II or with Her2 positive (P=0.023, P=0.041). Consistent with the KNSL4 mRNA expression, Kid protein expression was higher in cancer cells in situ than in normal tissues, but down-regulated in invasive carcinoma cells. In the KNSL4 transcription silenced subclone cell MDA-MB-435S/KNSL4 siRNA, cell proliferation capacity decreased, proportion of G2/M phase of the cell cycle increaced, and the mitotic block was observed. However, changes of capacity of clone formation, invasion and migration change in vitro and in vivo were not observed. Cell division cycle 25 homolog C (CDC25C) and activating transcription factor 7 interacting protein (ATF7IP) were validated as target genes negatively regulated by Kid.Conclusions: KNSL4 expression is an indicator of metastasis of breast cancer cells but not a regulator in metastasis progress. Kid plays a key role and may form a negative feedback loop with CDC25C in cell mitosis, and its abnormality may cause the cell malignant transformation and proliferation. KNSL4 expression may serves as molecular marker of the prognostic prediction and antitumor molecular target for breast cancer patients.